Rhodium-catalyzed hydroformylation of ketal-masked ß-isophorone: computational explanation for the unexpected reaction evolution of the tertiary Rh-alkyl via an exocyclic ß-elimination derivative.

Ketal-masked ß-isophorone (7,9,9-trimethyl-1,4-dioxaspiro[4.5]dec-7-ene) is an interesting case study of Rh-catalyzed hydroformylations not only for the competition between secondary and tertiary rhodium alkyls but also for the unexpected isomerization of the tertiary Rh-alkyl to the exocyclic olefin which undergoes hydroformylation, yielding the acetaldehyde derivative (2) of 7,9,9-trimethyl-1,4-dioxaspiro[4.5]decane. Although experimental results at 100 °C pointed to reaction reversibility, the reason for this kind of behavior was however obscure. A thorough density functional theory (DFT) computational investigation of the various transition states (TS) and intermediates along the reaction pathways making use of B3P86 hybrid functionals and the 6-31G* basis set, coupled to effective core potentials for Rh in the LanL2DZ valence basis set, has been carried out to shed some light on the reaction mechanism. The TS barrier heights, based on alkyl-Rh TS free energies, computed under the hypothesis of nonreversibility were in favor of a normal hydroformylation reaction (III:II = 70:30). While the endocyclic olefins produced skew or twisted arrangements of the six-membered ring similarly to the CO insertion TS that can be even higher than the alkyl-Rh ones, grid-point calculations during the potential energy surface (PES) scan produced the much more stable chair conformation for the exocyclic olefin complex. The relevant TS were found to be very favorable as well, thus explaining the preference for the exocyclic arrangement of the tertiary intermediate, for which the reaction is therefore entirely reversible and invariably proceeds to the acetaldehyde derivative (2). Conversely for the secondary isomers, the reaction is only partially reversible, thus enriching the tertiary fraction and producing the secondary aldehyde (3) in a very limited amount.

Computational prediction of selectivities in nonreversible and reversible hydroformylation reactions catalyzed by unmodified rhodium-carbonyls.

The regio- and stereoselectivities of the hydroformylation reaction catalyzed by an unmodified Rh catalyst have been investigated at the B3P86/6-31G* level with Rh described by effective core potentials in the LANL2DZ valence basis set for a number of either mono- or (1,1-, 1,2-, 1,3-) di-substituted substrates and compared with a variety of earlier results of ours, supplemented with free energy results when not already available. The computational prediction of regio- and stereoselectivities in nonreversible hydroformylations performed under mild reaction conditions is seemingly possible provided a careful conformational search for TS structures is carried out and all the low energy conformers are taken into account. The internal energy can be used to compute both the regio- and stereoselectivities in the hydroformylation of 1,1- and 1,3-substituted substrates with satisfactory results, whereas for 1,2-substituted substrates the regioselectivity determined from the internal energy is in good agreement with the experiment in the case of aliphatic olefins just for the lowest terms in the series (i.e., methyl and ethyl substituents), while the ratios are only qualitatively correct for the slightly bulkier iso-propyl and tert-butyl moieties. The theory/experiment agreement becomes decidedly better using the free energy differences instead.

The catalytic effect of water on the keto-enol tautomerism. Pyruvate and acetylacetone: a computational challenge.

The catalytic effect of explicit water molecules on the keto-enol tautomerism in a system of biological interest (enolpyruvate) has been investigated at the B3LYP/6-31++G** level by exploring the potential energy surface in the presence of 1 or 2 water molecules and comparing the energy profiles to the direct tautomerisation one. The water-assisted mechanisms turned out to be more favourable than the direct ones, in agreement with what occurred for the acetylacetone tautomerism in the presence of a single water molecule. To compare the behaviour of water in pyruvate and in acetylacetone, the two-water-assisted mechanism has been also examined for the latter at the B3LYP/6-31G* level. A number of stationary points for both compounds in vacuo and in the presence of explicit water molecules have been computed with DFT coupled to larger basis sets and at the MP2 level. Two diketo forms more stable than any of the keto-enol tautomers have been located, while for pyruvate the keto form is always more favourable than the enol one. The equilibrium constant for acetylacetone tautomerisation has been computed with high accuracy, performing a complete basis set extrapolation for the relative internal energy, to determine whether the quality of the method/basis set is responsible for the earlier modest agreement with the experimental value. Monte Carlo and molecular dynamics simulations helped characterize the solution structure and association features in the 0.03-0.22 molar concentration range.

Antioxidant properties of pterocarpans through their copper(II) coordination ability. A DFT study in vacuo and in aqueous solution.

The antioxidant activity of 3,9-dimethoxy-4-prenylpterocarpan (bitucarpin A) and 3,9-dihydroxy-4,8-diprenylpterocarpan (erybraedin C) is supposed to be related to their copper coordination ability. Therefore several complexes with Cu(2+) of low-energy conformers of these two prenylated pterocarpans, whose conformational landscape was the subject of a prior B3LYP/6-31G* study (Alagona, Ghio, Monti Phys. Chem. Chem. Phys. 2004, 6, 2849), have been taken into account at the same computational level, with the metal ion described by effective core potentials in the LanL2DZ valence basis set. Their metal ion affinity (MIA) values have been determined and compared with the results obtained earlier with the same methods for the preferred binding sites of plicatin B, a prenylchalcone that can exist in E and Z configurations as well as in tautomeric forms. The stability order of the metalated species at the various coordination sites strongly depends on their position and nature. The spin density of the cation upon ligand coordination becomes vanishingly small, whereas the ligand spin density approaches 1. Thus the ligand is oxidized to a radical cation (Ligand(*+)), while Cu(II) is reduced to Cu(I). A very favorable MIA is obtained in vacuo when Cu(2+) is chelated between the prenyl and O lone pair moieties for both pterocarpans (MIA = 370 and 380 kcal/mol for bitucarpin A and erybraedin C, respectively). High affinity values are found also when the cation is sequestered within the two end groups (prenyl pi density and D ring) in the O(t) configuration (MIA = 371 and 373 kcal/mol for bitucarpin A and erybraedin C, respectively). In aqueous solution, the solvent effect dampens the free energy differences and reduces the MIA especially when the ion is remarkably exposed to the solvent. Conversely, when Cu(2+) is sequestered, the MIA decrease in solution is limited (MIA = 327 and 360 kcal/mol for bitucarpin A and erybraedin C, respectively). The solvent effect is significantly larger in plicatin B, where the MIA is lowered by 80 to 140 kcal/mol, probably because (a) the screening ability of the substituted phenolic ring is lower and (b) the positive charge on the ligand is less efficiently delocalized than in the four fused ring system of pterocarpans.

Plicatin B conformational landscape and affinity to copper (I and II) metal cations. A DFT study.

The conformational landscape of the prenylchalcone plicatin B and some of its tautomers has been investigated at the B3LYP/6-31G* level in analogy to prior studies of ours on two structurally related prenylated pterocarpans. Since the antioxidant activity of these natural compounds is supposed to be related to their copper chelation ability, several complexes with Cu(i) and Cu(ii) metal cations, Cu(+) and Cu(2+), have been taken into account with the metal ions described by effective core potentials in the LANL2DZ valence basis set. The preferred binding sites on low-energy conformers of E and Z plicatin B have been determined and their metal ion affinity (MIA) values have been compared. Both cations give stable complexes with plicatin B, but the stability order of the metallated species at the various coordination sites strongly depends on the cation nature. In particular, for the E configuration the most stable Cu(+)-plicatin B ground-state structure features the metal cation bridged between the hydroxy O lone pairs and the prenyl pi density, while in the most stable Cu(2+) complex the cation is coordinated with the inner lone pairs of the oxygens in the methyl ester moiety bearing an anti methyl group. For the Z configuration, in contrast, the most stable Cu(2+) complexes are found with the metal ions dentated between the Z ester side chain and the prenyl pi density, while Cu(+) in addition is close to the aromatic ring density as well. A comparison of the Cu(+) and Cu(2+) affinity values demonstrates however that the affinity to Cu(2+) is decidedly much higher (by a factor of 3-4, depending on the arrangement type) than that to Cu(+), even including the possible B3LYP overestimate of the Cu(2+) binding energy with respect to BHLYP. A tentative evaluation of MIA in aqueous solution using the polarizable continuum model of the solvent shows a remarkable decrease for Cu(ii).

Computational prediction of the regio- and diastereoselectivity in a rhodium-catalyzed hydroformylation/cyclization domino process.

The regioselectivity of the hydroformylation reaction of 2-methyl-3-(3-acetylpyrrol-1-yl)prop-1-ene catalyzed by an unmodified Rh catalyst has been investigated at the B3LYP/6-31G* level with Rh described by effective core potentials in the LANL2DZ valence basis set. Considering the population of all the H-Rh(CO)3-olefin transition state complexes, a regioselectivity ratio (B:L) of 12:88 has been obtained, in satisfactory agreement with the experiment producing the chiral linear aldehyde as the only product. The aldehyde, after complete diastereoselective cyclization, yields a 1:1 mixture of 1-acetyl-6R(S)-methyl-8R(S)-hydroxy-5,6,7,8-tetrahydroindolizine (having the same configuration on both stereogenic carbon atoms) and 2-acetyl-6-methyl-5,6-dihydroindolizine [Lett Org Chem (2006) 3:10-12]. The reason for such a high degree of diastereoselectivity has been elucidated examining the B3LYP/6-31G* potential energy surface for the reactions leading to the RR and RS diastereomers on a model system (without the acetyl substituent) and the actual compound. In the absence of a catalyst, a very high barrier is found along the reaction pathway, whereas spontaneous annulation occurs to a protonated pentahydroindolizine in the presence of H+. When a counterion (F-) is added, the proton on the newly formed tetrahedral carbon is abstracted, obtaining a structure closer to the final product (tetrahydroindolizine). Replacing H+ with Rh+, an initial adduct along the RS path much more favorable than any of those computed along the RR one is located because of the presence of the acetyl group. Tentative approaching paths obtained using [Rh(CO)3]+, bound to the aldehyde O, feature a higher barrier along the RS one, and offer a convincing explanation for the observed diastereoselectivity.

Caco-2 cell permeability modelling: a neural network coupled genetic algorithm approach.

The ability to cross the intestinal cell membrane is a fundamental prerequisite of a drug compound. However, the experimental measurement of such an important property is a costly and highly time consuming step of the drug development process because it is necessary to synthesize the compound first. Therefore, in silico modelling of intestinal absorption, which can be carried out at very early stages of drug design, is an appealing alternative procedure which is based mainly on multivariate statistical analysis such as partial least squares (PLS) and neural networks (NN). Our implementation of neural network models for the prediction of intestinal absorption is based on the correlation of Caco-2 cell apparent permeability (P (app)) values, as a measure of intestinal absorption, to the structures of two different data sets of drug candidates. Several molecular descriptors of the compounds were calculated and the optimal subsets were selected using a genetic algorithm; therefore, the method was indicated as Genetic Algorithm-Neural Network (GA-NN). A methodology combining a genetic algorithm search with neural network analysis applied to the modelling of Caco-2 P (app) has never been presented before, although the two procedures have been already employed separately. Moreover, we provide new Caco-2 cell permeability measurements for more than two hundred compounds. Interestingly, the selected descriptors show to possess physico-chemical connotations which are in excellent accordance with the well known relevant molecular properties involved in the cellular membrane permeation phenomenon: hydrophilicity, hydrogen bonding propensity, hydrophobicity and molecular size. The predictive ability of the models, although rather good for a preliminary study, is somewhat affected by the poor precision of the experimental Caco-2 measurements. Finally, the generalization ability of one model was checked on an external test set not derived from the data sets used to build the models. The result obtained is of interesting practical application and underlines that the successful model construction is strictly dependent on the structural space representation of the data set used for model development.

Theoretical Investigation of Tautomeric Equilibria for Isonicotinic Acid, 4-Pyridone, and Acetylacetone in Vacuo and in Solution.

Tautomeric equilibria have been theoretically calculated for isonicotinic acid (neutral and zwitterionic forms), the 4-pyridone/4-hydroxypyridine system, and the keto-enol transformation for acetylacetone in vacuo and in tetrahydrofuran, methanol, and water solvents. Solvent, basis set, and cavity model effects have been studied in the integral equation formalism for the polarizable continuum model (IEF-PCM)/B3LYP framework, as well as the effect of the procedure, CHELPG or RESP, applied in fitting atomic charges to the in-solution molecular electrostatic potential (ELPO). The in-solution optimized geometries obtained at the IEF-PCM/B3LYP/6-31G* and 6-311++G** levels differ moderately but deviate from their gas-phase counterparts. Atomic charges fitted to the in-solution ELPO show small variations in the considered solvents, as well as when the united-atom cavity model, or a model with explicit consideration of polar hydrogens and scaled Bondi radii, has been applied. In contrast, the fitting procedure considerably affects the derived charges producing more separated atomic charges when the CHELPG rather than the RESP procedure is utilized. The fitted charges increase up to 20% in absolute value when the basis set is enlarged from 6-31G* to 6-311++G** in the IEF-PCM/B3LYP calculations. The relative free energy, calculated as ΔGtot = ΔEint + ΔG(solv) + ΔGthermal + (symmetry correction), in an ab initio/density funtional theory (DFT) + free energy perturbation (FEP)/Monte Carlo (MC) approximation strongly depends on the accepted value for the relative internal energy, ΔEint, of the tautomers. ΔEint is to be calculated at the IEF-PCM/QCISD(T)/cc-pVTZ//IEF-PCM/B3LYP/6-31G* level for the isonicotinic acid tautomers for producing relative free energies in aqueous solution close to experimental values. In other solvents, for this system and for the other two tautomeric equilibria, calculation of ΔEint at the IEF-PCM/B3LYP/6-31G* level produces ΔGtot in agreement up to 1 kcal/mol with the experimental values. FEP/MC ΔG(solv) calculations provide robust results with RESP charges derived by a fit to the in-solution ELPO generated at the IEF-PCM/B3LYP/6-31G* level. Molecular dynamics simulations pointed out that isonicotinic acid forms a dimeric zwitterion in tetrahydrofuran, in contrast to what happens in aqueous solution, and this structural peculiarity was interpreted as the reason for the failure of the ab initio/DFT + FEP/MC method in this particular solution.

Conformational landscape of (R,R)-pterocarpans with biological activity in vacuo and in aqueous solution (PCM and/or water clusters).

All possible combinations of stable dihedral values have been considered in vacuo at the B3LYP/6-31G level for 3,9-dihydroxy-4,8-diprenylpterocarpan (erybraedin C), whose hydroxy out-out conformation had been examined earlier together with the conformational preferences of 3,9-dimethoxy-4-prenylpterocarpan (bitucarpin A) at the same level (Phys. Chem. Chem. Phys. 2004, 6, 2849). The structure with O5 trans with respect to H6a (O(t)) is about 2 kcal/mol less stable in vacuo than that with one of the H6 trans to it (H(t)); in aqueous solution its energy gap is nearly conserved. The in-in arrangement of the hydroxyl groups of erybraedin turns out to be preferred in vacuo (even considering zero point and thermal effects), where pseudo H-bonds are formed between hydroxy hydrogens and pi electron distributions of prenyl groups. The continuum solvent effect (water) at the IEF-PCM/B3LYP/6-31G level on the relative stability of the various rotamers is very limited both on bitucarpin and erybraedin. Considering the dihydrated derivatives, significant differences in the solvation energy are found between the distinct hydration sites, increasing in the order: methoxy O, ring O, hydroxy O, and hydroxy H. In hydroxy-water interactions, in fact, water prefers to behave as an H-bond acceptor unless nearby bulky groups prevent its approach. Interestingly enough, a bridging water molecule between the hydroxy H of erybraedin and the prenyl group can be found. The inclusion of BSSE corrections in hydroxy-water interactions decidedly favors out-out hydrated arrangements, followed by out-in and in-out ones. Bulk solvent effects with IEF-PCM about the dihydrated systems almost invert the stability order found in vacuo. When a four-water cluster is considered using QM methods, waters gather in H-bonded pairs around the solute OH groups. MD simulations, carried out on a pterocarpan solute (J. Phys. Chem. B 2005, 109, 16918), supply water adducts consistent with a liquid state that have also been embedded in the continuum solvent.

Structure and dynamics of the hydrogen-bond network around (R,R)-pterocarpans with biological activity in aqueous solution.

Molecular dynamics simulations were carried out in the presence of 2380 water molecules (TIP3P) to explore the conformational preferences of 3,9-dimethoxy-4-prenylpterocarpan (bitucarpin A) and 3,9-dihydroxy-4,8-diprenylpterocarpan (erybraedin C) and the H-bond network around them, using the empirical general AMBER force field (GAFF). Specific angle and torsional parameters have been improved in order to match the geometries of the minimum energy structures obtained from an earlier DFT/ab initio study in vacuo, taking into account a few configurations [Alagona, G.; Ghio, C.; Monti, S. Phys. Chem. Chem. Phys. 2004, 6, 2849-2857]. RESP partial charges were assigned to reproduce the electrostatic potential determined at the HF/6-31G level of theory. The analysis of trajectories allowed the conformations of bitucarpin and erybraedin as well as the distribution of water molecules around them to be elucidated. During one of the simulations only, the scaffold of erybraedin undergoes interconversion from its most stable Ht conformation to the Ot one and vice versa. Radial distribution functions, coordination numbers, and angular distributions put forward the extent of solvent structure and the hydrogen bonding behavior of their various (methoxy, hydroxyl, or ethereal) oxygen atoms. The distribution of solvent molecules in the first and second solvation shells as well as the residence times for the different solute-solvent interacting sites have been considered.

Theoretical Conformational Analysis for Neurotransmitters in the Gas Phase and in Aqueous Solution. Serotonin.

Conformational analyses have been performed for protonated serotonin in the gas phase, aqueous solution, and in the binding cavity of a 5-HT2A receptor model. DFT geometry optimizations have been performed in the gas phase at the B3LYP/6-31G* levels. Optimized calculations up to the B3LYP/6-311++G** level find two low-energy gauche conformations separated by 8-10 kcal/mol barriers from a trans conformation with relative energy of about 6 kcal/mol. In aqueous solution as concluded from IEF-PCM/B3LYP/6-31G* and IEF-PCM/MP2/6-31G*//IEF-PCM/B3LYP/6-31G* continuum solvent calculations as well as Monte Carlo free energy perturbation simulations with explicit solvent molecules, those barriers decrease to 2-7 kcal/mol, while the two gauche and one trans conformers are within a 3 kcal/mol relative free energy range. The solute is strongly hydrated by about three water molecules around the -NH3(+) group and by one water molecule for each of the pyrrole and phenolic hydrogen atoms. Docking studies of the protonated ligand predicted both gauche and trans ligand conformers to favorably interact with the 5-HT2A receptor in its hypothesized binding cavity. The theoretical studies confirm the experimental results regarding strong interactions with the Asp155 and Ser159 residues (TM helix III) and the interactions of the indole ring with Phe, Trp, and Tyr side chains in TM V, VI, and VII helices within a 24 kcal/mol range for the relative interaction energies.

Quantum mechanical study of stereoselectivity in the oxazaborolidine-catalyzed reduction of acetophenone.

Chiral oxazaborolidines, known as CBS catalysts after the work of Corey, Bakshi and Shibata, are used for the stereoselective reduction of prochiral ketones to secondary chiral alcohols. Due to their relative low cost, ease of use, and high selectivity, their popularity has remarkably grown in the last 15 years. Oxazaborolidine-catalyzed reductions have been much studied, both experimentally and computationally, by means of semiempirical methods. Though, a more accurate high level quantum mechanical study on the complete system, capable of elucidating reliably the origins of stereoselectivity, is still lacking. Therefore, the acetophenone (PhMK) reduction with Corey's oxazaborolidine has been modeled for the first time with ab initio and DFT-B3LYP calculations on the complete system as well as with AM1. Calculations on the complexation of BH(3) to CBS, which can occur only in a cis fashion with respect to the hydrogen on the stereogenic C-4 carbon atom, have allowed us to confirm the great rigidity of Corey's catalyst, possibly determining its excellent enantioselectivity. Acetophenone-CBS-BH(3) complexes were characterized at various levels of theory, and it was found that the picture obtained depends heavily on the method adopted. A computational strategy for identifying the hydride transfer transition states of the competing pathways was developed and tested, using a model system for which the transition state geometry was already known. The application of the TS search method to the reduction of acetophenone allowed the characterization of the TS's for the competing pathways in this reaction, making it possible to predict with good quantitative accuracy the stereochemical outcome of the reaction at all the levels of theory adopted. The characterization of the intermediate oxazadiboretane products confirmed that the highly exothermic hydride transfer provides the thermodynamical drive for the reaction.

Cholic acid derivatives containing both 2-naphthylcarbamate and 3,5-dinitrophenylcarbamate groups: a combined circular dichroism-molecular mechanics approach to the definition of their molecular conformation.

CD spectra of the chiral auxiliaries for enantioselective HPLC N-allyl-N'-methyl-3,12-bis(2-naphthyl)carbamoyloxy-7-(3,5-dinitrophenyl)carbamoyloxycholan-24-amide (1), N-allyl-N'-methyl-3-(3,5-dinitrophenyl)carbamoyloxy-7,12-bis(2-naphthyl)carbamoyloxycholan-24-amide (2), N-allyl-N'-methyl-3,7-bis(2-naphthyl)carbamoyloxy-12-(3,5-dinitrophenyl)carbamoyloxycholan-24-amide (3), and N-allyl-N'-methyl-3,7,12-tris(2-naphthyl)carbamoyloxycholan-24-amide (4) are presented. To determine the preferred conformations of those chiral auxiliaries, a random search based on the aromatic side-chain conformational degrees of freedom was performed and the energy was minimized using two different molecular mechanics force fields. The low energy structures presenting common features were arranged in groups and selected exploiting appropriate filters. The calculation of theoretical CD spectra according to the De Voe model has allowed a further discrimination among the conformations, specifying which of them gave calculated CD spectra in acceptable agreement with the experimental ones. Finally, taking into account the additivity of the contributions of each 2-naphthylcarbamate chromophore to the CD spectrum of the cholic acid derivatives, and, hence, choosing, for derivatives 1-3, those conformations in which the 2-naphthylcarbamate groups take a similar disposition as in 4, the preferentially assumed conformation of each compound was obtained. A molecular dynamics simulation in the presence of acetonitrile allowed the fluctuations of one of the structures, used as a test case, depending on environmental effects, to be examined.

Theoretical conformational analysis for neurotransmitters in the gas phase and in aqueous solution. Norepinephrine.

The natural neurotransmitter (R)-norepinephrine takes the monocationic form in 93% abundance at the physiological tissue pH of 7.4. Ab initio and DFT/B3LYP calculations were performed for 12 protonated conformers of (R)-norepinephrine in the gas phase with geometry optimizations up to the MP2/6-311++G level, and with single-point calculations up to the QCISD(T) level at the HF/6-31G-optimized geometries. Four monohydrates were studied at the MP2/6-31G//HF/6-31G level. In the gas phase, the G1 conformer is the most stable with phenyl.NH(3)(+) gauche and HO(alc).NH(3)(+) gauche arrangements. A strained intramolecular hydrogen bond was found for conformers (G1 and T) with close NH(3)(+) and OH groups. Upon rotation of the NH(3)(+) group as a whole unit about the C(beta)-C(alpha) axis, a 3-fold potential was calculated with free energies for barriers of 3-12 kcal/mol at the HF/6-31G level. Only small deviations were found in MP2/6-311++G single-point calculations. A 2-fold potential was calculated for the phenyl rotation with free energies of 11-13 kcal/mol for the barriers at T = 310 K and p = 1 atm. A molecular mechanics docking study of (R)-norepinephrine in a model binding pocket of the beta-adrenergic receptor shows that the ligand takes a conformation close to the T(3) arrangement. The effect of aqueous solvation was considered by the free energy perturbation method implemented in Monte Carlo simulations. There are 4-5 strongly bound water molecules in hydrogen bonds to the conformers. Although hydration stabilizes mostly the G2 form with gauche phenyl.NH(3)(+) arrangement and a water-exposed NH(3)(+) group, the conformer population becomes T > G1 > G2, in agreement with the PMR spectroscopy measurements by Solmajer et al. (Z. Naturforsch. 1983, 38c, 758). Solvent effects reduce the free energies for barriers to 3-6 and 9-12 kcal/mol for rotations about the C(beta)-C(alpha) and the C(1)(ring)-C(beta) axes, respectively.

The intramolecular mechanism for the second proton transfer in triosephosphate isomerase (TIM): a QM/FE approach.

The intramolecular mechanism we earlier proposed [Alagona, G.; Desmeules, P.; Ghio, C.; Kollman, P. A. J Am Chem Soc 1984, 106, 3623] for the second proton transfer of the reaction catalyzed by triosephosphate isomerase (TIM) is examined ab initio at the HF and MP2/6-31+G** levels in vacuo for two conformers of the enediolate phosphate (ENEP), with the ethereal oxygen of the phosphate group either syn (X), as in the crystal structure, or anti (Y) with respect to the enediolate carbonyl O. The barrier height for the intramolecular proton transfer occurring in enediolate is very sensitive to electron correlation corrections. The MP2 internal energy barrier is much lower than the HF one, while the free energy (FE) barrier is even more favorable, indicating that the enzyme presence is not requested to speed up that step. An investigation of the dynamical aspects of the mechanism, along the pathway from ENEP A (with H on O(1)) to TS and from TS to ENEP B (with H on O(2)), was, however, carried out in the presence of the enzyme field while using a neutral His-95 with its proton on Ndelta. To perform the FE simulations, it was necessary to parametrize in the AMBER force-field the ENEP A, TS and B species, whose partial charges have been determined with the RESP procedure, with the X and Y arrangements of the phosphate head. Actually, the FE/QM approach produced a low barrier and a substantial balance between A and B, especially at the MP2 level. The trajectories, analyzed paying a particular attention to the positions assumed by His-95 and by the other active site residues, put forward somewhat different H-bond patterns around the X or Y enediolate phosphate.